Research Interests

RNA molecules are uniquely capable of encoding and controlling the expression of genetic information, often as a consequence of their three-dimensional structures. We are interested in understanding RNA-mediated initiation of protein synthesis, and RNA-protein complexes involved in targeting proteins for export out of cells. We are also investigating the early steps in gene regulation by RNA interference.

Current Projects

The role of mRNA structure in miRNA-mediated gene regulation

MicroRNAs (miRNAs) regulate endogenous eukaryotic genes by repressing gene expression through direct base-pairing interactions with their target messenger RNAs (mRNAS). Although the principles governing miRNA-mRNA interactions are critical to understanding the mechanisms of miRNA-mediated gene regulation, they remain incomplete. To date, the rules used to predict miRNA-mRNA interactions have been based on one-dimensional sequence analysis. A more complete picture of miRNA-mRNA interactions should take into account the ability of RNA to form two- and three-dimensional structures. We are investigating the role of mRNA structure in the efficiency and specificity of targeting by miRNAs. Specifically, we are investigating the structure of Alu elements found within the 3′ UTRs of many human mRNAs and whether these structured domains serve as targets of a subset of human miRNAs. We are using both in vitro biochemical methods and cell-based assays to probe the relationship between miRNA binding and mRNA structure.

Internal Ribosome Entry Site (IRES) RNAs

Most eukaryotic and viral messages initiate translation by a mechanism involving recognition of a 7-methylguanosine cap at the 5' end of the mRNA. In a few cases, however, translation occurs via a cap-independent mechanism in which an Internal Ribosome Entry Site (IRES) in the 5' untranslated region of the mRNA recruits the ribosome. In Hepatitis C virus (HCV), the ~400 nucleotide IRES folds into a magnesium-dependent structure in which loops thought to interact with the ribosome are exposed on the surface of the RNA. Point mutations that destroy IRES activity disrupt the folded structure of the RNA. The IRES is formed from two independently folding structural domains. One of these, the "core", binds specifically to the 40S subunit of eukaryotic ribosomes, while the other domain interacts with initiation factor eIF3. Structures of the IRES-40S subunit complex, determined by cryo-electron microscopy, revealed that the IRES induces a significant conformational change in the 40S subunit upon binding. This conformational change helps lock the start of the viral mRNA protein coding sequence into the correct site on the 40S subunit.

We determined structures of the HCV IRES in complex with the human translational machinery using cryo-EM, showing how the IRES can functionally replace proteins that help position most cellular mRNAs on the ribosome. Using affinity purified samples, mass spectrometry has revealed the full composition and post-translational modification states of IRES-bound complexes that assemble in human cell extracts, and shown how the HCV IRES induces assembly of active human 80S ribosomes.

Current work strives to uncover the structure and function of all domains of the HCV IRES, and their interactions with participants of the human translational machinery such as the 40S subunit and eIF3, which are both required for efficient translation initiation by the IRES. In addition to structural and biochemical approaches and to address these questions, we are very interested in elucidating the mechanism of action of small molecule inhibitors of the HCV IRES. Such compounds could both make an important contribution to the field’s search for new drugs to treat Hepatitis C, and also reveal more subtle requirements for IRES function than can be determined from mutational analysis alone. In addition to using mechanistic biochemistry to look at the function of putative small molecule inhibitors from the literature, we have also undertaken our own high-throughput screen to try to find new small molecules that specifically inhibit translation driven by the HCV IRES.

Structural and biochemical characterization of a CRISPR-mediated bacterial immune system

Prokaryotes have evolved a nucleic acid-based immune system that shares some functional similarities with RNA interference in eukaryotes. Central to this system are DNA repeats called CRISPRs (*C*lustered *R*egularly *I*nterspaced *S*hort *P*alindromic *R*epeats). CRISPRs are genetic elements containing direct repeats separated by unique spacers, many of which are identical to sequences found in phage and other foreign genetic elements. Recent work has demonstrated the role of CRISPRs in adaptive immunity and shown that small RNAs derived from CRISPRs (crRNAs) are implemented as homing oligos for the targeted interference of foreign DNA.

Phylogenetic analysis of CRISPR-associated (Cas) proteins suggests there are at least seven distinct versions of this immune system. We have initiated genetic, biochemical and structural studies aimed at understanding the function of each of the Cas proteins associated with the immune system from *Pseudomonas aeruginosa* PA14. We have recently published the 2.2Å resolution crystal structure of the Pa14 Cas1 protein, revealing a distinct fold and a conserved divalent metal ion-binding site. Cas1, a metal-dependent, DNA-specific endonuclease, is the sole universal protein marker of the CRISPR-mediated immune system. Current work in the lab focuses on biochemical and structural characterization of proteins and protein/RNA complexes involved in crRNA biogenesis and foreign DNA targeting.

Structure and Mechanism of the Signal Recognition Particle (SRP)

The signal recognition particle (SRP) is a highly conserved
ribonucleoprotein responsible for transport of nascent polypeptides
targeted for secretion or membrane insertion. In prokaryotes, the SRP
consists of one protein (Ffh) and one RNA molecule (4.5S RNA), and both
are required for SRP activity. The RNA sequence corresponding to the
Ffh binding site has been maintained through evolution, and is
virtually identical in organisms from the three kingdoms of life -
bacteria, archaea and eukaryotes. The RNA plays a key, yet
undetermined, role in the protein targeting pathway. In 2000 we
determined the crystal
structure of the complex at 1.5 Å resolution, revealing a
fascinating network of contacts at the RNA-protein interface that
explain the observed evolutionary conservation. Using site-directed
hydroxyl radical probing, we discovered that the association of the SRP
with its receptor triggers a dramatic conformational change in the
complex, localizing the SRP RNA and the adjacent signal peptide binding
site at the SRP-receptor heterodimer interface. The orientation of the
RNA explains how peptide binding and GTP hydrolysis can be coupled
through direct structural contact during cycles of SRP-directed protein
translocation. Recent experiments show that the position of the SRP RNA
within the SRP-receptor complex enhances the rate of GTP hydrolysis in
the complex above a critical threshold required in vivo.
Work towards a crystal structure of the SRP-receptor complex (a ~130 kD
assembly) has been aided by the selection and purification of multiple
antibody proteins using phage display technology.

RNA Recognition by Dicer Enzymes

Double-stranded RNA induces potent and specific gene silencing in a broad range of eukaryotic organisms. This mode of gene silencing, called RNA interference (RNAi), acts at the transcriptional level through formation of heterochromatin and at the post-transcriptional level through mRNA degradation and translational suppression. In all cases, RNAi begins with the processing of endogenous or introduced precursor RNA into micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) 21-25 nucleotides in length by the enzyme Dicer. We recently solved the crystal structure of an intact Dicer enzyme, revealing how Dicer functions as a molecular ruler to measure and cleave duplex RNAs of a specific length. The structure has now been refined to higher resolution, and a series of mutant forms of Dicer have been used to delineate the roles of various domains and interactions both in vitro and in vivo. Ongoing work focuses on determining how Dicer interacts with other components of the RNAi pathway and how diced RNAs are targeted to specific mRNAs.